580
A. Singhal et al.
the biomolecule as compared to the bulk solution (Mohamad et al. 2015). Physical methods for immobilization include entrapment, adsorption, and microencapsulation. Chemical strategies cover various ways of covalently linking well-defined
functional groups and include the use of affinity ligands such as biotin-streptavidin.
Compared to their free forms, immobilized enzymes are usually more stable and
easier to handle (Homaei et al. 2013). Further, the corresponding reaction products
can be separated easily and is of special relevance to the food and pharmaceutical
industries (Kirk et al. 2002). Nevertheless, the structural disruption of biomolecules
due to immobilization poses a substantial challenge. For example, irreversible deactivation of enzymes upon covalent immobilization renders both the enzymes and
often-costly support unusable. Similarly, non-covalent attachment of biomolecules
is fraught with leaching in aqueous media. Cross-linking in gel matrix or carrier
can result in a reduction in activity due to misfolding. Further, the preparation of
cross-linked enzyme aggregates necessitates the crystallization of enzymes, which
is a laborious and unpredictable process (Sheldon and van Pelt 2013). Site-specific
conjugation approaches have witnessed significant advances in recent years. The
sophisticated application of organic chemistry and molecular biology has enabled
the anchoring of enzymes and antibodies on various supports thereby expanding the
scope of protein microarrays, biosensors and flow reactor systems (Wallace and Balskus 2014). In this context, nucleic acid based scaffolds and conjugation approaches
have occupied a distinctive niche. The predictable and programmable self-assembly
characteristics of DNA and RNA have been exploited for the design and construction
of complex nanostructures that facilitate the detailed spatial organization of attached
moieties (Zhang et al. 2014).
Nucleic acid scaffolds have been used for organizing enzyme cascade reactions.
Double-stranded DNA templates result in only modest improvements in enzyme
performance (Muller and Niemeyer 2008; Niemeyer et al. 2002). Small DNA nanostructures have been more effective in this regard and several novel designs including double-crossover tiles (Fu et al. 2014) and three-point star DNA (Rothemund
2006) are noteworthy (Fig. 2). Scaffolded DNA origami templates have provided a
unique opportunity for precise spatial anchoring of biomolecules. 3D DNA origami
templates have been deployed for eliciting superior enzyme cascade efficiencies
when compared to freely diffusing enzymes in solution (Fu et al. 2012). The semiconfined character of rectangular origami systems has been partially implicated in
such improved enzyme efficiency along with the stabilizing effect of the origami.
Other strategies for enzyme immobilization on nucleic acid scaffolds include the use
of nucleic acid binding protein domains (Nakata et al. 2015) and aptamer binding
interactions (Hasegawa et al. 2016).
Developments in the nucleic acid based scaffolding and conjugation of
biomolecules should be juxtaposed with the advances in DNA-based biosensors. For
example, electrochemical DNA (E-DNA) sensors have gained traction and have been
used to detect the presence of hazardous substances such as drugs and heavy metal
ions (Lubin and Plaxco 2010). E-DNA sensors rely on the ability of the constituent
nucleic acid to make contact with an electrode generating detectable Faradaic current
in response to a specific analyte (Lubin and Plaxco 2010). Nucleic acid secondary
A. Singhal et al.
the biomolecule as compared to the bulk solution (Mohamad et al. 2015). Physical methods for immobilization include entrapment, adsorption, and microencapsulation. Chemical strategies cover various ways of covalently linking well-defined
functional groups and include the use of affinity ligands such as biotin-streptavidin.
Compared to their free forms, immobilized enzymes are usually more stable and
easier to handle (Homaei et al. 2013). Further, the corresponding reaction products
can be separated easily and is of special relevance to the food and pharmaceutical
industries (Kirk et al. 2002). Nevertheless, the structural disruption of biomolecules
due to immobilization poses a substantial challenge. For example, irreversible deactivation of enzymes upon covalent immobilization renders both the enzymes and
often-costly support unusable. Similarly, non-covalent attachment of biomolecules
is fraught with leaching in aqueous media. Cross-linking in gel matrix or carrier
can result in a reduction in activity due to misfolding. Further, the preparation of
cross-linked enzyme aggregates necessitates the crystallization of enzymes, which
is a laborious and unpredictable process (Sheldon and van Pelt 2013). Site-specific
conjugation approaches have witnessed significant advances in recent years. The
sophisticated application of organic chemistry and molecular biology has enabled
the anchoring of enzymes and antibodies on various supports thereby expanding the
scope of protein microarrays, biosensors and flow reactor systems (Wallace and Balskus 2014). In this context, nucleic acid based scaffolds and conjugation approaches
have occupied a distinctive niche. The predictable and programmable self-assembly
characteristics of DNA and RNA have been exploited for the design and construction
of complex nanostructures that facilitate the detailed spatial organization of attached
moieties (Zhang et al. 2014).
Nucleic acid scaffolds have been used for organizing enzyme cascade reactions.
Double-stranded DNA templates result in only modest improvements in enzyme
performance (Muller and Niemeyer 2008; Niemeyer et al. 2002). Small DNA nanostructures have been more effective in this regard and several novel designs including double-crossover tiles (Fu et al. 2014) and three-point star DNA (Rothemund
2006) are noteworthy (Fig. 2). Scaffolded DNA origami templates have provided a
unique opportunity for precise spatial anchoring of biomolecules. 3D DNA origami
templates have been deployed for eliciting superior enzyme cascade efficiencies
when compared to freely diffusing enzymes in solution (Fu et al. 2012). The semiconfined character of rectangular origami systems has been partially implicated in
such improved enzyme efficiency along with the stabilizing effect of the origami.
Other strategies for enzyme immobilization on nucleic acid scaffolds include the use
of nucleic acid binding protein domains (Nakata et al. 2015) and aptamer binding
interactions (Hasegawa et al. 2016).
Developments in the nucleic acid based scaffolding and conjugation of
biomolecules should be juxtaposed with the advances in DNA-based biosensors. For
example, electrochemical DNA (E-DNA) sensors have gained traction and have been
used to detect the presence of hazardous substances such as drugs and heavy metal
ions (Lubin and Plaxco 2010). E-DNA sensors rely on the ability of the constituent
nucleic acid to make contact with an electrode generating detectable Faradaic current
in response to a specific analyte (Lubin and Plaxco 2010). Nucleic acid secondary